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Cloning of the 18S rDNA sequence from arachidis, the causal pathogen of groundnut scab

Chuan Tang Wang*, Xiu Zhen Wang, Yue Yi Tang, Dian Xu Chen, Jian Cheng Zhang, Feng Gao Cui and Shan Lin Yu Shandong Research Institute (SPRI), Qingdao 266100, PR China *Corresponding author: [email protected]

Citation: Wang CT, Wang XZ, Tang YY, Chen DX, Zhang JC, Cui FG and Yu SL. 2009. Cloning of the 18S rDNA sequence from , the causal pathogen of groundnut scab. Journal of SAT Agricultural Research 7.

Introduction Materials and methods

Historically, groundnut scab caused by Sphaceloma A PCR (polymerase chain reaction) template was arachidis Bit. & Jenk. was reported from Argentina, prepared from diseased groundnut tissue of leaflets or Brazil, Colombia and Japan (Frison et al. 1990, Xu stems using a highly simplified protocol for groundnut 2009). Now it has become a great threat to groundnut published by us earlier in this year with minor (Arachis hypogaea) production in south China (Zhang et modification (Wang et al. 2009). Briefly, the unused end al. 1995, Hu et al. 2000, Fang et al. 2006, Wang et al. (without a tip) of a ball point pen refill was utilized as a 2006). In recent years, the disease is gaining increasing hole puncher, and the discs (6.5 mm2) were then smashed importance in north China, including Shandong province, using a thin-walled PCR tube mounted with a 1 ml pipette the leading groundnut producer of China (Figs. 1 and 2). tip as a pestle, in 40 μl of 0.25 M sodium hydroxide While the pathogen is thought to be solely restricted to (NaOH) in an eppendorf microcentrifuge tube. The the Arachis, Emechebe (1980) found that mixture was boiled for 30 sec. Then 160 μl of 100 mM Sphaceloma species also attacked cowpea (Vigna Tris-HCl (pH 7.6) with 5 mg ml-1 of polyvinylpyrrolidone unguiculuta), causing symptoms similar to groundnut (PVP) was added and the mixture was boiled for 2 min. scab. After centrifugation at 10,000 rpm for 5 min, 90 μl of the The study is intended to clone the 18S rDNA sequence supernatant was collected and placed into an eppendorf from S. arachidis and provide some useful information tube with 450 μl of TE buffer. The DNA template thus for the of the pathogen.

Figure 1. Diseased plants of groundnut scab (left) and less affected plants (right) in a field in Laixi, Shandong, China. Figure 2. Close-up view of symptoms of groundnut scab (note sinuous branches and coalescence of lesions).

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prepared holds good at 4°C for a week and at −20°C for The PCR products were recovered using the EZNA several months. Cycle Pure Kit (Omega Bio-Tek, Inc.), and were ligated The universal primers for fungal 18S rDNA, viz, into a PBS-T vector. Competent Escherichia coli cells NS26 (5’-CTGCCCTATCAACTTTCGA-3’) and R518 (DH5a) were used in heat-shock transformation. White (5’-ATTACCGCGGCTGCTGG-3’), were used in PCR colonies were picked up, checking presence of the inserts amplification (Zhang et al. 2007). PCR was conducted in using colony PCR prior to sequencing. DNA sequencing a 50 μl reaction volume containing 1 μl of PCR template was performed on an ABI 3730XL DNA sequencer using (about 2 ng), 25 μl of Tiangen 2× Taq platinum the T3/T7 primer. The DNA sequences obtained were MasterMix (Tiangen Biotech) and 2 μl of each primer compared with sequences deposited in GenBank using (10 μM). DNA amplification was performed in a blastn. Phylogenetic tree was constructed using MEGA Biometra Tgradient Thermocycler (Biometra Ltd) using version 4 (Tamura et al. 2007). the following thermal cycling profile: one cycle at 95°C for 6 min to activate Taq platinum, followed by 32 cycles at 94°C for 1 min, at 55°C for 50 s, and at 72°C for 1 min, Results and discussion with a final extension step at 72°C for 6 min. The PCR products of expected size (near 300 bp) were obtained and diseased tissue from leaflets or stems gave the same result (Fig. 3). As expected, no PCR product was obtained from healthy groundnut tissue. Totally 7 cloned samples were sequenced, and multiple sequence alignment using Lasergene 7.1.0 (DNAStar Inc.) revealed that all of these insert sequences had no difference. The sequence is shown in Figure 4. The taxonomic status of S. arachidis is still not clear. Presently, the pathogen is tentatively placed in Deuteromycota. Molecular data may be of some utility in taxonomic studies; however, unfortunately, no nucleic acid information regarding the pathogen is available. A neighbor-joining tree (Fig. 5) and a minimum evolution tree were constructed based on the 18S rDNA sequences of S. arachidis and related fungal species. Both the trees showed similar topology. The results indicated that the pathogen was in close relationship with Basidiomycetes species.

Acknowledgment. Financial support from the earmarked fund for Modern Agro-industry Technology Research System (MATRS) Peanut Program (Grant No. Figure 3. Amplification products of the fungal 18S rDNA ntcytx-19), Ministry of Agriculture, China, China Natural sequence (M: Tiangen 100 bp DNA ladder; L: Diseased tissue Science Foundation (Grant No. 30300224) and 863 New from a leaflet; S: Diseased tissue from a stem). and High Technology Project (Grant No. 2006AA10A114) is gratefully acknowledged.

Figure 4. 18S rDNA sequence of Sphaceloma arachidis.

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Figure 5. A bootstrap neighbor-joining tree based on 18S rDNA sequences of Sphaceloma arachidis and related sequences from GenBank. (Note: Numbers on branches represent the bootstrap values.)

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